Brazina Jan, Svadlenka Jan, Macurek Libor, Andera Ladislav, Hodny Zdenek, Bartek Jiri, Hanzlikova Hana
a Department of Cell Signaling and Apoptosis.
Cell Cycle. 2015;14(3):375-87. doi: 10.4161/15384101.2014.988019.
Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression.
死亡结构域相关蛋白6（DAXX）是一种组蛋白伴侣，是细胞凋亡和转录的假定调节因子，也是DNA损伤后p53介导的基因表达的候选调节因子。DAXX在DNA损伤时会发生磷酸化，然而这种修饰的调控及其与p53的关系仍不清楚。在这里，我们表明，在暴露于电离辐射或基因毒性药物依托泊苷和新制癌菌素的人类细胞中，DAXX以ATM激酶依赖性方式迅速磷酸化。我们的缺失和定点诱变实验确定丝氨酸564（S564）是DAXX的主要ATM靶向位点，免疫荧光实验揭示了S564磷酸化的DAXX定位于PML核体。此外，使用一组人类细胞类型，我们确定p53调节的Wip1蛋白磷酸酶是体外和细胞内DAXX在S564处磷酸化的关键负调节因子。与Wip1新出现的致癌作用一致，其使DAXX去磷酸化的影响在具有功能获得性突变和/或过表达Wip1的癌细胞系中最为明显。出乎意料的是，虽然Wip1缺失在DNA损伤前后均增加了DAXX磷酸化，并增加了p53的稳定性和转录活性，但敲低DAXX既不影响p53的稳定性，也不影响p53介导的Gadd45a、Noxa、Mdm2、p21、Puma、Sesn2、Tigar或Wip1的表达。一致地，对通过基因编辑、TALEN介导的DAXX缺失的细胞分析证实了这样一种观点，即DNA损伤时p53介导的基因表达既不需要磷酸化的DAXX也不需要非磷酸化的DAXX。总体而言，我们确定ATM激酶和Wip1磷酸酶是DAXX-S564磷酸化的相反调节因子，并提出DAXX磷酸化的作用以及DAXX本身与p53介导的基因表达无关。
